Antiproliferative effects of D-allose associated with reduced cell division frequency in glioblastoma

Recent studies have shown that D-allose, a rare sugar, elicits antitumor effects on different types of solid cancers, such as hepatocellular carcinoma, non-small-cell lung cancer, and squamous cell carcinoma of the head and neck. In this study, we examined the effects of D-allose on the proliferation of human glioblastoma (GBM) cell lines (i.e., U251MG and U87MG) in vitro and in vivo and the underlying mechanisms. D-allose treatment inhibited the proliferation of U251MG and U87MG cells in a dose-dependent manner (3–50 mM). However, D-allose treatment did not affect cell cycles or apoptosis in these cells but significantly decreased the cell division frequency in both GBM cell lines. In a subcutaneous U87MG cell xenograft model, intraperitoneal injection of D-allose (100 mg/kg/day) significantly reduced the tumor volume in 28 days. These data indicate that D-allose-induced reduction in cell proliferation is associated with a subsequent decrease in the number of cell divisions, independent of cell-cycle arrest and apoptosis. Thus, D-allose could be an attractive additive to therapeutic strategies for GBM.


D-allose suppresses tumor growth in GBM xenograft mice
We prepared 18 subcutaneously U87MG-injected xenograft mice and treated them with D-allose.After 21 days, a significant difference was observed between the two groups (control: 659 ± 238 mm 3 vs.D-allose: 450 ± 117 mm 3 ; P = 0.03, n = 9 for each group), and these differences gradually increased each day.The experiment was terminated when an obvious difference was observed in tumor growth on the 28th day (2791 ± 727 mm 3 vs.1700 ± 796 mm 3 ; P = 8.0 × 10 −6 , n = 9 for each group) (Fig. 2a).The mice were anesthetized (Fig. 2b), and the resected tumors appeared smaller in the D-allose-treated group than in the control group (Fig. 2c).A significant difference in the weight of the resected tumors was observed (Fig. 2d).No mouse was eliminated because of excessive tumor growth.The body weight of the mice was very similar between the two groups (27.8 ± 1.8 g vs. 26.7 ± 1.3 g; P = 0.27, n = 9 for each group) (Fig. 2e).These results suggest that D-allose has antitumor effects on GBM in vivo.In contrast, no differences in the HE and immunohistochemically stained sections with anti-Ki67 and anti-ɤH2AX antibody were observed (Fig. 2f).This suggests that D-allose does not suppress the ratio of proliferating cells or affect DNA damage.

D-allose has no observable effects on cell-cycle progression and apoptosis in GBM cells
U251MG and U87MG cells were cultured with or without 50-mM D-glucose or D-allose and were harvested, and their nuclei were stained with propidium iodide (PI).The percentage of cells in each cell-cycle phase was analyzed using flow cytometry.After 24 h of treatment, no observable difference was observed among the treatment groups (Fig. 3a, b).The proportions of cells in each phase were analyzed.In U251MG cells, no significant changes were observed in the proportion of cells in the G0/1 phase between the control (72.3% ± 2.2%), D-glucose (70.5% ± 4.2%; P = 0.40, n = 4), and D-allose treatment groups (70.7% ± 3.6%; P = 0.40, n = 4) (Fig. 3c).Conversely, in U87MG cells, the proportion tended to increase in D-allose-treated cells (77.9 ± 4.2%) compared with that in the control group (74.9% ± 4.4%) (P = 0.056, n = 4) (Fig. 3d).Because the proportion tended to increase in D-glucose-treated cells (78.0% ± 2.9%) (P = 0.067, n = 4), this tendency may not be unique to D-allose.Moreover, the results were similar when the treatment time was 96 h (data were not shown).These data suggest that D-allose did not affect the cell cycle in GBM cells.Flow cytometry was used to evaluate apoptosis in U251MG and U87MG cells.FITC-labeled annexin V and PI were used to identify apoptotic cells.In principle, cells in early apoptosis are positive for annexin V but negative for PI, and cells in late apoptosis or dead cells are positive for both annexin V and PI.Therefore, cells considered viable are negative for both annexin V and PI.In both GBM cell lines, no difference in the population of apoptotic cells was observed between cells treated with D-allose and those treated with equimolar D-glucose after 72 h (Fig. 4a, b).The proportion of cells in late apoptosis was analyzed.In U251MG cells, the mean proportion was 39.3% ± 12.8% for the control group, 36.9% ± 7.0% for the 50-mM D-glucose treatment group (P = 0.64, n = 3), and 42.7% ± 6.0% for the 50-mM D-allose treatment group (P = 0.52, n = 3), and no significant difference was observed between the groups (Fig. 4c).Conversely, in U87MG cells, the proportion tended to increase in the D-allose treatment group (4.2% ± 1.2%) compared with that in the control group (2.1% ± 0.4%) (P = 0.075, n = 4) (Fig. 4d).Furthermore, because the proportion also tended to increase in the 50-mM D-glucose treatment group (4.7% ± 2.2%) (P = 0.12, n = 4), this tendency may not be unique to D-allose.The results were similar when the treatment time was changed from 24 to 96 h, and therefore, we could not observe any apoptotic changes in GBM cells following D-allose treatment.

D-allose reduces cell division frequency in GBM cells
Flow cytometry assay was performed to evaluate the cell division frequency in U251MG and U87MG cells using CytoTell™ Ultragreen.As stained cells divide, this reagent is equally distributed between daughter cells and can  www.nature.com/scientificreports/be measured as a successive halving of the fluorescence intensity of the dye.A difference in fluorescence intensity between any groups suggests that the cell division frequency differs between the groups; therefore, the higher the intensity, the lower the cell division frequency.GBM cells were stained with CytoTell™ Ultragreen and cultured for 96 h to clarify these differences.Representative data showed that in D-allose-treated cells, the convexity of the graph shifted toward the higher side with differences in cell population appearance (Fig. 5a, b).These data have revealed that D-allose treatment significantly reduced the cell division frequency in U251MG cells (the mean relative fluorescence intensity was 1.00 for the control group, 1.30 ± 0.11 for the 50-mM D-glucose treatment group [P = 0.039, n = 3], and 1.82 ± 0.19 for the 50-mM D-allose treatment group [P = 0.017, n = 3]) and U87MG cells (the mean relative fluorescence intensity was 1.00 for the control group, 1.02 ± 0.20 for the 50-mM D-glucose treatment group [P = 0.86, n = 4], and 2.15 ± 0.51 for the 50-mM D-allose treatment group [P = 0.02, n = 4]) (Fig. 5c, d).Significant differences were observed between D-glucose-and D-allose-treated U251MG cells (P = 0.012) and U87MG cells (P = 0.0075).

Discussion
Our in vitro and in vivo data revealed that D-allose treatment suppressed the proliferation of GBM cells.However, D-allose did not affect the cell cycle or apoptosis in these cells.Interestingly, D-allose treatment reduced the cell division frequency in GBM cell lines.Collectively, these data suggest that D-allose elicits antitumor effects through mechanisms independent of cell cycle arrest and apoptosis.
D-allose significantly inhibited cell proliferation in GBM cell lines (both U251MG and U87MG) in a dosedependent manner.Similar concentration-dependent effects of D-allose were observed in head and neck squamous cell carcinoma 17 , ovarian cancer 11 , and hepatocellular carcinoma 12 cells.Consistent with in vivo data with other cancers 14,15,[17][18][19] , this study showed that GBM tumor growth was significantly inhibited by D-allose, which reduced the tumor volume to 60.9%.Thus, these data indicate that D-allose inhibits GBM cell proliferation.Moreover, the amount of D-allose administered intraperitoneally in this in vivo study, when converted to human body weight and glucose, is equivalent to 100 mL of 5% glucose solution once a day, which we thought would be easy to apply clinically.
Several mechanisms underlying the antitumor effects of D-allose have been reported.In ovarian cancer, Sui et al. 11 observed that approximately 8% ± 3% of apoptotic cells were identified in cells treated with D-allose (50 mM/120 h).In human head and neck carcinoma, Mitani et al. 16 observed moderate apoptosis induction in Ca9-22 cells treated with D-allose (50 mM for 72 h).Similarly, Indo et al. 17  www.nature.com/scientificreports/for 48 h) did not induce HuH-7 apoptosis, a hepatocellular carcinoma cell line.In this study, apoptosis was evaluated using flow cytometry; however, we did not observe any apoptotic changes in D-allose-treated cells.In U87MG cells, the proportion of cells in late apoptosis tended to increase compared with that in the control group; however, these changes were not statistically significant.Furthermore, the number of apoptotic cells tended to increase even in glucose-treated cells.Therefore, this tendency of D-allose is not unique and may be due to osmotic pressure or other effects.Thus, D-allose treatment (50 mM for 72 h) did not induce apoptosis in GBM cells.These data suggest that D-allose-induced cell apoptosis is not consistently induced in several cancer cell lines.Several reports have revealed that D-allose induces cell-cycle arrest in several cancer cell lines.D-allose induces G1 12 and S 16 phase arrest, which was observed in head and neck cancer and hepatocellular carcinoma, respectively.Conversely, D-allose-induced G2/M arrest has been reported in head and neck cancer, non-smallcell lung cancer, and ovarian cancer 11,14,17 .However, no cell-cycle arrest was observed in D-allose-treated cells.In U87MG cells, the G0/1 phase tended to increase; however, this change was not statistically significant.These data combined with those of apoptosis support the notion that cellular responses (e.g., changes in cell cycles and induction of apoptosis) to D-allose are inconsistent in various cancer cells 16 .Moreover, the results of this study suggest that the antitumor effects of D-allose on GBM cells are mediated by an alternative mechanism.
To confirm the effects of D-allose on cell proliferation, the cell division frequency was investigated using the label-retaining method.Data showed that D-allose treatment significantly reduced the number of cell divisions compared with that in the control and D-glucose treatment groups.These data combined with those of other experiments indicated that D-allose decreases the number of cell divisions, which are not mediated by cell-cycle arrest or cell apoptosis.Based on these data, we speculate that D-allose uniformly decelerates the entire cell cycle but does not arrest the cells in any specific cell-cycle phase.The results obtained in vivo, that is, no changes in pathological findings despite a significant decrease in tumor volume, are consistent with this hypothesis.However, note that the effects of D-allose might be different from those on widely known "slow-cycling cells, " which means a population of cells with an innately slow cell cycle or a population that has slowed its cell cycle to survive an unexpected environment, characterized by chemoresistance or tumor relapse in oncology 21,22 .In these cells, cell proliferation inhibition is thought to be due to the prolonged non-cycling state of each cell 22 .Therefore, our observation of a slow cell cycle is slightly different from that of slow-cycling cells.Therefore, further careful and detailed studies should be conducted in the future.
This study is the first to examine the effects of rare sugars on brain tumors.The rare sugar D-allose was found to have an antitumor effect on tumors arising from the brain, which is one of the most sugar-consuming organs in the body.This study has several limitations.First, the molecular mechanism responsible for the antitumor effects of D-allose remains unclear.Second, the brain uptake of D-allose is lower than that of D-glucose in healthy mice but is higher in ischemia-disrupted brains than in healthy brains 23 .Therefore, whether D-allose can be disrupted into brain tumors remains unclear, and this study did not examine intra-tumor D-allose.Third, only the U87MG cell line was used in the in vivo study.We attempted to use the U251MG cell line in an in vivo study; however, these cells failed to grow in nude mice.Fourth, combination therapies with radiotherapy or chemotherapy have not been investigated.Future studies should be conducted to address these issues.
This study demonstrated that D-allose treatment reduces the proliferation of GBM cells.Our data also suggest that D-allose decelerates the cell cycle through a mechanism that is independent of cell apoptosis and cell-cycle arrest.Further studies are required to determine the detailed molecular mechanism responsible for the antitumor effects of D-allose on brain tumors.

Cell proliferation assay
The water-soluble tetrazolium (WST)-1 assay was used to assess the proliferation ability of both cell lines following the manufacturer's instructions (Takara Bio).
Briefly, cells were seeded in a 24-well plate and incubated until confluence reached approximately 30-40%.The cells were serum starved for 24 h, the medium was removed, and a fresh medium containing variant concentrations (0, 3, 5, 10, 30, and 50 mM) of D-glucose or D-allose was added.After a subsequent 48-h incubation, 50-µL WST-1 reagent was added in addition to the 500-µL cell culture medium in each well, and the mixture was incubated for 2 h at 37 °C.Finally, absorbance was measured at 450 and 690 nm using a microplate reader (Corona Electric Co.).The value was obtained by subtracting the absorbance at 690 nm from that at 450 nm.D-glucose was used as a control for equivalent sugar osmotic pressure.

Figure 1 .
Figure 1.Antiproliferative effects of D-allose on human GBM cell lines and toxicity against healthy fibroblast cells in vitro.The graph shows the results of the WST-1assay of various D-allose treatments in GBM cell lines (a) and healthy fibroblast cells (b).Tests were performed on U251 (n = 12 in each concentration), U87MG (n = 12 in each concentration), and MEF cells (n = 12 in each concentration).Comparisons were also performed with D-glucose-treated samples at the same concentration.Statistically significant (P < 0.01, P < 0.001) reductions in cell proliferation compared with that in the control or D-glucose-treated samples are represented by asterisks (*, **).GBM, glioblastoma; WST-1, water-soluble tetrazolium-1; MEF, mouse embryonic fibroblast.

Figure 2 .Figure 3 .
Figure 2. Antitumor effects of D-allose on human GBM cell lines in vivo.Eighteen subcutaneous tumor model mice of U87MG were divided into the control and D-allose groups.Each of them was administered with natural saline or D-allose solution intraperitoneally daily.(a) The graph shows tumor volume changes over time in each group.(b) A picture showing mice with tumors.(c) A picture showing resected tumors on the 28 th day.(d) The graph shows the weight of the resected tumors in each group.€ The graph shows changes in body weight over time.(f) Representative images show HE and immunohistochemistry staining for Ki67 and ɤH2AX.Statistically significant (P < 0.05, P < 0.01) decreases in tumor volume compared with that in the control group are represented by asterisks (*, **)(Bar = 100 µm).GBM, glioblastoma; HE, hematoxylin and eosin.

Figure 4 .
Figure 4. Apoptosis analysis of U251MG and U87MG cells.Cells were stained with FITC-labeled annexin V and PI, and the intensity of fluorescence was assessed at 525 nm (FITC) and 675 nm (PI).Representative data of the flow cytometry assay of U251MG (a) and U87MG (b) cells treated with control, D-glucose, or D-allose are shown.The values in each graph indicate the cell ratios of cells in late apoptosis.(c, d) The graphs show the mean values of three experiments for each group.FITC, fluorescein isothiocyanate; PI, propidium iodide; ns, not significant.

Figure 5 .
Figure 5. Cell division analysis of U251MG and U87MG cells.Cells were stained with CytoTell™ Ultragreen solution, and the intensity of fluorescence was assessed at 519 nm.Representative data of the flow cytometry assay of U251MG (a) and U87MG (b) cells treated with control, D-glucose, or D-allose are shown.Each photo was taken just before assay (Bar = 100 µm).The graphs show the mean value of the relative fluorescence intensity to the control of three (U251MG, c) or four (U87MG, d) experiments.Statistically significant (P < 0.05, P < 0.01) reduction in the cell division frequency compared with that in the control or D-glucose-treated samples is represented by asterisks (*, **).GBM, glioblastoma. https://doi.org/10.1038/s41598-023-46796-4